Wolbachia Stimulates Immune Gene Expression and Inhibits Plasmodium Development in Anopheles gambiae

The over-replicating wMelPop strain of the endosymbiont Wolbachia pipientis has recently been shown to be capable of inducing immune upregulation and inhibition of pathogen transmission in Aedes aegypti mosquitoes. In order to examine whether comparable effects would be seen in the malaria vector Anopheles gambiae, transient somatic infections of wMelPop were created by intrathoracic inoculation. Upregulation of six selected immune genes was observed compared to controls, at least two of which (LRIM1 and TEP1) influence the development of malaria parasites. A stably infected An. gambiae cell line also showed increased expression of malaria-related immune genes. Highly significant reductions in Plasmodium infection intensity were observed in the wMelPop-infected cohort, and using gene knockdown, evidence for the role of TEP1 in this phenotype was obtained. Comparing the levels of upregulation in somatic and stably inherited wMelPop infections in Ae. aegypti revealed that levels of upregulation were lower in the somatic infections than in the stably transinfected line; inhibition of development of Brugia filarial nematodes was nevertheless observed in the somatic wMelPop infected females. Thus we consider that the effects observed in An. gambiae are also likely to be more pronounced if stably inherited wMelPop transinfections can be created, and that somatic infections of Wolbachia provide a useful model for examining effects on pathogen development or dissemination. The data are discussed with respect to the comparative effects on malaria vectorial capacity of life shortening and direct inhibition of Plasmodium development that can be produced by Wolbachia

Commentaries on viewpoint : physiology and fast marathons

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Immune gene expression and challenges with <i>Brugia pahangi</i> in <i>Ae. aegypti</i> somatically infected with <i>w</i>MelPop, and effects of immune knockdown on <i>Wolbachia</i> density.

<p>A) The expression of four immune genes were analyzed by qRT-PCR: a peptidoglycan recognition protein, <i>PGRPS1</i>; cecropin D, <i>CECD</i>; CLIP-domain serine protease, <i>CLIPB37</i>; and a C-type galactose-specific lectin. Adult females were injected with <i>w</i>MelPop or the buffer alone, approximately seven days post-eclosion. RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition). B) The mean numbers of L3 stage (infective) larvae per mosquito are shown following <i>B. pahangi</i> challenge in <i>Ae. aegypti</i> Ref^m strain previously injected with <i>w</i>MelPop or buffer; * <i>P</i><0.05. Numbers above bars show the prevalence of filarial infection as a proportion of mosquitoes that contained at least one L3 <i>Brugia</i> larva over the total number of mosquitoes dissected in each category. C) We measured the levels <i>Wolbachia ftsZ</i> gene expression as a proxy for <i>Wolbachia</i> density and normalized the qRT-PCR data to the mosquito <i>Actin5C</i> gene. Two sets of three females per time point injected with either dsLacZ or dsRel2 were assayed. <i>ftsZ</i> gene expression was found to be higher in dsRel2-injected mosquitoes than in dsLacZ-injected mosquitoes at both six and ten days post injection. The mean level of <i>Rel2</i> transcript in dsRel2-injected mosquitoes was confirmed to be approximately 40% of that in dsLacZ injected mosquitoes at both time points. These data suggest that the immune effectors controlled by the Imd pathway (<i>Rel2</i>-controlled) can influence <i>Wolbachia</i> densities.</p

Oligonucleotide primers used in quantitative PCR experiments and dsRNA synthesis.

<p>Previously published oligonucleotides are indicated by the reference number following the gene name.</p

Immune gene expression in <i>An. gambiae</i> somatically infected with <i>w</i>MelPop.

<p>The expression of six immune genes were analyzed by qRT-PCR: leucine-rich repeat immune protein, <i>LRIM1</i>; thioester-containing protein, <i>TEP1</i>; cecropin, CEC1; defensin, <i>DEF1</i>; C-type lectin, <i>CTL4</i>; and clip-domain serine protease, <i>CLIPB3</i>. Adult <i>An. gambiae</i> females were injected with <i>E. coli</i>, <i>w</i>MelPop or the buffer alone, 2–3 days post-eclosion, and RNA was extracted from these adults eight days after injection. Expression was normalized to non-injected adult females of the same age from the same colony. Error bars show the SEM of three biological replicates, each containing eight adult females (total of 24 mosquitoes per condition).</p

Model of possible effects of <i>w</i>MelPop on malaria vectorial capacity.

<p>Vectorial capacity is a measure that describes the transmission potential of a mosquito population and is independent of <i>Plasmodium</i> prevalence. It can be thought of as proportional to the number of infectious bites that occur per day after a single infectious human arrives in a previously malaria-free area. If we assume recruitment to the adult mosquito stage is constant then vectorial capacity can be written (<i>A b</i> (1−<i>μ</i>)^τ)/<i>μ</i> where <i>b</i> is the ability of the mosquito to transmit <i>Plasmodium</i>, <i>μ</i> is adult daily survival, <i>τ</i> is the length of the intrinsic incubation period of the <i>Plasmodium</i> and all other parameters are combined in <i>A </i><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001143#ppat.1001143-Smith1" target="_blank">[42]</a>. The figure plots vectorial capacity as transmission (<i>b</i>) and daily survival (<i>μ</i>) are each reduced because of the presence of <i>Wolbachia</i> by a multiplicative factor (1−<i>x</i>) where <i>x</i> varies in the range 0 to 1 (parameters: <i>b</i> = 1; <i>μ</i> = 0.1; <i>τ</i> = 1; <i>A</i> = 1). A more advanced analysis tailored to a specific system might want to include age-specific adult mortality, the effect of <i>Wolbachia</i> on mosquito population dynamics and seasonality.</p

Immune gene expression in the <i>An. gambiae w</i>MelPop-infected MOS55 cell line.

<p>The expression of six immune genes as described for <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1001143#ppat-1001143-g001" target="_blank">Figure 1</a> were analyzed by qRT-PCR, for the <i>An. gambiae</i> MOS55 cell culture infected with <i>w</i>MelPop, normalized to expression of these genes in a tetracycline treated, <i>w</i>MelPop free, genetically identical, MOS55 cell culture. Three samples of cells were taken from the cultures at different times; error bars show the SEM of these three samples.</p

Increased Reward-Related Behaviors during Sleep and Wakefulness in Sleepwalking and Idiopathic Nightmares

We previously suggested that abnormal sleep behaviors, i.e., as found in parasomnias, may often be the expression of increased activity of the reward system during sleep. Because nightmares and sleepwalking predominate during REM and NREM sleep respectively, we tested here whether exploratory excitability, a waking personality trait reflecting high activity within the mesolimbic dopaminergic (ML-DA) system, may be associated with specific changes in REM and NREM sleep patterns in these two sleep disorders